An ever-enlarging world population has increased demands on water resources worldwide. Indeed, this population increase is directly proportional to the potential for surface and ground water contamination by pathogenic organisms associated with increased waste burdens. To ensure good public health, there is a need for readily available methods to detect and enumerate pathogens in water. Unfortunately, despite years of testing and research, no single procedure is available for the reliable detection of the major waterborne pathogens. Indeed, there are no standardized methods for detecting all of these pathogens. The methods that are available are usually time-consuming and expensive.
Routine or periodic monitoring of water for the presence of pathogens is essential in situations such as wastewater reclamation, during and after waterborne outbreaks, and for water sources with a frequent history of contamination. This is largely due to the observation that most enteric pathogens appear intermittently and in low concentrations in aquatic environments. Thus, potentially pathogenic organisms may be present in a water supply and go undetected, largely due to their low numbers and the limitations of current testing methods, including relatively low sensitivity levels.
Despite advances in public health technology, water and food remain important reservoirs of diarrheal and other diseases of humans and other animals. Infectious represents a significant public health concern. According to one estimate, infectious diarrhea results in the hospitalization of 200,000 children in the United States each year, at an annual cost of one billion dollars (M. Ho et al., "Rotavirus as a cause of diarrheal morbidity and mortality in the United States," J. Infect. Dis., 158:1112-1116, 1988).
Worldwide, waterborne disease is of even greater significance, with over 250 million reported cases of waterborne disease and more than 10 million deaths annually (J. D. Snyder and M. H. Merson, "The magnitude of the global problem of acute diarrheal disease: a review of active surveillance data," Bull. World Health Organ., 60:605-613 [1982]). When other sources of diarrheal disease are taken into consideration the figures are even more staggering, with these diseases claiming the lives of over 5 million children per year in developing countries (T. L. Hale, "Genetic basis of virulence in Shigella species," Microbiol. Rev., 55:206-224 [1991]).
Most of the cases of waterborne diarrheal disease result from the contamination of drinking water supplies with human fecal material. Contamination of ground water in local areas may occur through such mechanisms as seepage of sewage into aquifers and by improperly developed or poorly protected wells. When factors such as recreational exposure to contaminated salt and fresh water are also taken into consideration, diarrheal disease takes on even greater importance.
Various infectious agents are associated with human waterborne diseases, including Campylobacter, E. coli, Leptospira, Pasteurella, Salmonella, Shigella, Vibrio, Yersinia, Proteus, Giardia, Entoamoeba, Cryptosporidium, hepatitis A virus, Norwalk, parvovirus, polio virus, and rotavirus. Worldwide, the most common bacterial diarrheal diseases are associated with waterborne transmission of Shigella, Salmonella, pathogenic E. coli, Campylobacter jejuni, and Vibrio cholerae (Singh and McFeters, "Detection methods for waterborne pathogens," pp. 125-156, in R. Mitchell (ed.), Environmental Microbiology, [Wiley-Liss, New York, 1992]). Table 1 lists important characteristics of diseases associated with a few of the most significant organisms.
TABLE 1 __________________________________________________________________________ Waterborne Diarrheal Bacterial Diseases Most Commonly Reported Incubation Organism Disease Period Common Symptoms __________________________________________________________________________ Shigella sp. Shigellosis 1-7 days Diarrhea, fever, cramps, tenesmus, dysentery* Salmonella typhimurium Salmonellosis 6-72 hours Abdominal pain, diarrhea, nausea, vomiting, fever S. typhi Typhoid fever 1-3 days Abdominal pain, fever, chills, diarrhea or constipation, intestinal hemorrhage Pathogenic E. coli Diarrhea 12-72 hours Diarrhea, fever, vomiting Campylobacter jejuni Gastroenteritis 1-7 days Abdominal pain suggesting acute appendicitis, fever, headache, malaise, diarrhea, vomiting Proteus sp. Scombroid fish Few minutes Headache, dizziness, vomiting, nausea, poisoning to 1 hour peppery taste, burning throat, facial swelling and flushing, stomach pain, itching Yersinia enterocolitica Yersiniosis 24-36 hours Severe abdominal pain, fever, headache Vibrio parahaemo-lyticus 12 hours Vomiting, diarrhea, abdominal pain, fever Vibrio cholerae Gastroenteritis 1-3 days Vomiting, diarrhea, dehydration __________________________________________________________________________
Swimming-associated outbreaks caused by Shigella, Giardia, Norwalk-like viruses, and other enteroviruses have been well documented [See e.g., Makintubee et al., "Shigellosis outbreak associated with swimming," Am. J. Public Health 77:166-168 [1987]; F. J. Sorvillo et al., Shigellosis associated with recreational water contact in Los Angeles County," Am. J. Trop. Med. Hyg., 38:613-617[1988]).
The following table lists the majority of waterborne infectious bacteria which are associated with human diarrheal and non-diarrheal disease.
TABLE 2 ______________________________________ Infectious Bacteria Transmitted by Water Commonly Associated Organism Diseases in Humans ______________________________________ Acinetobacter calcoaceticus Nosocomial infections Aeromonas hydrophila Enteritis, A. sobria wound infections A. caviae Campylobacter jejuni Enteritis C. coli Chromobacterium violaceum Enteritis Citrobacter spp. Nosocomial infections Clostridium perfringens, type C Enteritis Enterobacter spp. Nosocomial infections E. coli, various serotypes Enteritis Flavobacterium meninogsepticum Nosocomial infections, meningitis Francisella tularensis Tularemia Fusobacterium necrophorum Liver abscesses Klebsiella spp. Nosocomial infections, pneumonia Leptospira icterohaemorrahagia Leptospirosis and other Leptospira spp. Legionella pneumophila Legionellosis and other Legionella spp. Morganella morganii Urethritis, nosocomial infections Mycobacterium tuberculosis Tuberculosis M. marinum and other Mycobacterium spp. Granuloma, dermatitis Plesiomonas shigelloides Enteritis Pseudomonas pseudomallei Melioidosis Pseudomanas spp. Dermatitis, ear infections Salmonella enteritidis Enteritis S. montevideo B (salmonellosis) S. typhimurium and other Salmonella serotypes S. paratyphi A and B Paratyphoid fever S. typhi Typhoid fever Serratia marcesens Nosocomial infections Shigella spp. Dysentery Staphylococcus aureus Wounds, food poisoning Vibrio cholerae Cholera V. alginolyticus Enteritis V. fluvialis Wound infections V. mimicus V. parahaemolyticus V. vulnificus Other Vibrio spp. Yersinia enterocolitica Enteritis ______________________________________ *After T. C. Hazen and G. A. Toranzos, "Tropical Source Water," p. 33, in G. A. McFeters, Drinking Water Microbiology [SpringerVerlag, New York, 1990]).
While the presence of pathogens in drinking and recreational waters presents a significant public health concern, recovery of pathogens from environmental samples is generally difficult. In addition to the usually low numbers of organisms present in the water, nutrient limitations and environmental stressors produce unpredictable physiological and morphological changes in these pathogens. This makes their isolation and identification problematic. Organisms injured due to these environmental stressors often exhibit atypical reactions and require specialized handling for their resuscitation (see e.g., Singh and McFeters, p. 132-133).
Often, organisms are present but are unculturable (Id., at 131-159; see also, J. J. Byrd et al., "Viable but nonculturable bacteria in drinking water," Appl. Environ. Microbiol., 57:875-878 [1991]; C. Desmonts et al., "Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater," Appl. Environ. Microbiol., 56:1448-1442 [1990]; and J. J. Byrd and R. R. Colwell, "Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment," Appl. Environ. Microbiol., 56:2104-2107 [1990]). Unless other methods are used for their detection (e.g., immunoassays) these viable, but non-culturable organisms may present an undetected threat to public health.
In addition, the methods commonly used to detect these pathogens were initially designed for clinical, rather than environmental samples. This is of significance in view of the different ecological niche occupied by clinical as compared with environmental isolates. Clinical isolates are usually provided needed nutrients by their host animal and are generally protected from harsh environmental conditions such as cold, heat, damaging chemicals and radiation. In contrast, environmental isolates must deal with these environmental conditions and effectively compete with organisms naturally present and adapted to life in the environment. Pathogenic organisms are rarely readily adaptable to prolonged survival in the environment. Thus, "indicator" organisms are used as a prognostic indication of whether pathogens may be present in a particular sample.
Use of Indicator Organisms to Detect Fecal Contamination of Water
Problems associated with recovery of pathogens from water led to the development of methods to detect and enumerate "indicators" of fecal contamination. These organisms serve to indicate whether a given water supply is contaminated with fecal material, without actually testing for the presence of pathogens. This contamination is viewed as predictive of the potential presence of enteric pathogens (i.e., without the presence of fecal material, the chances of these pathogens being present is usually remote). However, a number of issues remain to be resolved, not the least of which is the significance of the presence of indicator organisms in water supplies.
Criteria for the establishment of the "ideal" indicator include the following factors: 1) the indicator should always be present in the presence of pathogens; 2) the indicator should always be present in a predicable ratio with pathogenic organisms; 3) the indicator should be specific for fecal contamination; 4) the indicator should be able to resist water treatment and disinfection processes to the same or a slightly greater extent than the pathogens; and 5) the indicator should be detectable by simple and rapid methods.
Historically, "coliforms" have served as the indicator bacteria for fecal contamination in United States water supplies. However, term "coliform" encompasses four genera (Escherichia, Citrobacter, Enterobacter, and Klebsiella); many of these species are commonly found in the environment in the absence of fecal contamination. Although all of these genera may be recovered from domestic sewage in large numbers, only E. coli is consistently and exclusively found in feces (see e.g., A. P. Dufour, "E. coli: the fecal coliform, in A. W. Hoadley and B. J. Dutka, Bacterial Indicators/Health Hazards Associated with Water, [ASTM, Philadelphia, 1976], p. 48). Thus, coliform detections methods are not specific for the determination of whether a water supply has been contaminated with fecal matter. Nonetheless, regulations based on detection and enumeration of "total coliforms" have been in effect in the United States since 1914 (i.e., the Treasury Department Standards of 1914; subsequent standards have been promulgated by the U.S. Public Health Service, and presently, by the US. Environmental Protection Agency [EPA]).
Recognition of the fact that most of the organisms included in the designation "total coliforms" are not of fecal origin, led to the development of tests to detect "fecal coliforms," for a subgroup of thermotolerant organisms included within the total coliforms. However, this designation is also not specific, as it includes E. coli, as well as various Klebsiella strains. Despite the fact that although there are substantial extra-fecal sources of Klebsiella, and this organism is infrequently found in human feces, the use of the "fecal coliform" designation and tests to identify these organisms remain routine (reviewed by V. J. Cabelli, Health Effects Criteria for Marine Recreational Waters, EPA-600/1-80-031, [August, 1983], pp.11-12).
Furthermore, the correlation between coliform densities in water and the incidence of waterbome disease originally postulated by Kehr and Butterfield in 1943 (R. W. Kehr and C. T. Butterfield, "Notes on the relationship between coliforms and enteric pathogens," Public Health Repts. 58:589-596 [1943]) have not been supported by experimental tests (Batik et al., "Routine monitoring and waterborne disease outbreaks," J. Environ. Health 45:227-230 [1984]). Quite simply, there has been no direct evidence presented that the level of coliform contamination correlates well with waterborne disease outbreaks (see Pipes, p. 434-435). Nonetheless, due to the lack of better methods, the detection of coliforms as indicator bacteria continues into the present.
Coliform detection may be accomplished by various methods, including multiple tube fermentation (i.e., most probable number or "MPN" determinations), membrane filtration, the "presence-absence" test, and various rapid enzyme (e.g., the MUG test) and immunoassay methods. Important considerations with these methods include the large time, equipment and personnel commitment necessary to conduct and interpret these tests.
COLIFORM DETECTION METHODS
Most Probable Number (MPN). The MPN method is a labor, time and supply intensive method, which involves three distinct stages of specimen processing (the presumptive (with lauryl tryptose broth), completed (with brilliant green lactose bile broth) and confirmed tests (with LES Endo or EMB). FIG. 1 illustrates the steps involved in MPN analysis for detection of coliforms. As is apparent from this figure, the MPN method requires 3-4 days in order to produce confirmatory results, and statistical analysis to quantitate the organisms present.
This procedure has been developed to separate organisms within the coliform group into "total" and "fecal" coliforms. Prior enrichment of organisms in a presumptive test medium is required for optimum recovery of fecal coliforms. These methods are used as confirmatory tests conducted with various selective media and elevated incubation temperatures (e.g., 44.5.degree. C.). Thus, there is also a significant time and labor commitment associated with these methods.
Membrane Filtration. In membrane filtration, a known volume of water sample is passed through a membrane filter which is then placed on growth media (e.g., M-Endo or LES-Endo), and incubated overnight. All colonies with characteristics common to coliforms are considered to be members of the coliform group. An advantage of membrane filtration is that preliminary results are usually available in 24 hours. However, verification of colony identification is recommended, usually requiring additional days in order to conduct the needed biochemical tests.
Additional disadvantages with the membrane filtration method include fouling of membranes with debris and suspended solids present in water. These particulates prevent free flow of water through the membrane, greatly slowing the process. In addition, the presence of particulate material on the membrane often interferes with organism growth, preventing reliable identification of bacteria. In this situation, reliable enumeration estimates are also precluded due to the presence of visible particulates present on the membrane which may be confused with colonies, the possibility that colonies are present under the particulate matter, yet not be visible for counting, and the potential interference with organism growth due to the composition of the particulates (e.g., the particulate may be comprised of a material toxic to the organisms).
Membrane filtration methods are especially unsuitable for use with "dirty" water. This is a significant consideration in many settings, especially testing of environmental waters.
Membrane Filtration Method Modifications. A seven hour fecal coliform test similar to the membrane filtration process has also been described. In this technique, the water sample is filtered and the filter placed on M-7 FC agar and incubated at 41.5.degree. C. [American Public Health Association-American Water Works Association-Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, 16th ed., [APHA, Washington, D.C.], 1985; hereinafter, "Standard Methods"]. Yellow colonies representing fecal coliforms are enumerated after seven hours of incubation. However, different growth rates of colonies necessitate a compromise between sensitivity of detection and enumeration. That is to say, because different organisms grow at different rates, some organisms will not have had sufficient time to produce visible colonies on the medium by the time enumeration is conducted.
The value of this test is perhaps questionable, in view of its deletion from the most recent edition of Standard Methods.
Another method developed by Reasoner, in conjunction with Geldrich [D. J. Reasoner and E. E. Geldreich, "Rapid detection of water-borne fecal coliforms by .sup.14 CO.sub.2 release," in A. N. Sharpe and D. S. Clark, (eds.) Mechanizing Microbiology, [Charles C. Thomas Publishers, 1978], pp. 120-139) involves concentration of bacteria on a membrane filter which is then placed in M-FC broth which contains radiolabelled .sup.14 C-mannitol. Major problems with these methods involve the use of radioactivity and the attendant disposal and handling concerns, as well as the need for specialized and expensive instruments. The tubes are incubated for 2 hours at 35.degree. C., followed by 2.5 hours at 44.5.degree.. Release of .sup.14 CO.sub.2 due to microbial metabolism is then assayed by liquid scintillation spectrometry.
An alternate radioactive test was developed by Dange et al. (V. Dange et al., "One hour portable test for drinking waters," Water Res., 22:133-137 [1988]). This method is based on the correlation of .sup.32 P uptake by organisms present in a water sample incubated in a synthetic medium. Thus, these methods require highly trained laboratory personnel and are not suitable for use in many labs.
Presence-Absence Test. The presence-absence test to detect the presence of coliforms involves the inoculation of broth with 100 ml samples of water, followed by incubation at 25.degree. C. for 24-48 hours. If acid and gas is produced in the medium, the test is positive for the presence of coliforms (see e.g., Standard Methods, at p. 882-884). No enumeration of organisms is attempted, nor are any identification methods utilized. Thus, the information garnered from this method is very limited.
Fluorometric and Enzymatic Tests. Detection methods for coliforms with fluorometric tests and numerous variations on the basic technology have also been developed. Other substrate-based methods include the use of such compounds as ortho-nitrophenyl-.beta.-D-galactopyranoside (ONPG) and 4-methylumbelliferyl-.beta.-D-glucuronide (MUG). These methods utilize fluorogenic or chromogenic substrates to detect coliform metabolism, as opposed to direct detection and enumeration of organisms. Thus, the only data available from these test methods relate to the presence or absence of organisms which possess the necessary enzymatic machinery to produce the detectable color compounds from a given substrate.
The MUG test is also problematic in that many clinically important E. coli strains are negative. For example, the highly virulent and very difficult to treat, E. coli 0157:H7 serotype associated with recent foodborne disease outbreaks is negative in this test (see e.g., E. W. Frampton and L. Restaino, "Methods for Escherichia coli identification in food, water and clinical samples based on beta-glucuronidase detection," J. Appl. Bacteriol., 74:223-233 [1993]). Indeed, there is a large proportion of .beta.-glucuronidase negative E. coli (see e.g., G. W. Chang et al., "Proportion of .beta.-D-glucuronidase-negative Escherichia coli in human fecal samples," Appl. Environ. Microbiol., 55:335-339 [1989]). Furthermore, species within other genera such as Staphylococcus, Streptococcus, Clostridium, and the anaerobic corynebacteria also produce .beta.-glucuronidase (Frampton and Restaino, p. 223). Thus, not only is the test not highly sensitive, it is not specific. These reports raise serious questions regarding the reliability of these testing methods.
Bacteriophages. In addition to culture and enzymatic detection methods, bacteriophages have also been used with some limited success as indicators of fecal contamination (R. S. Wensel et al., "Evaluation of coliphage detection as a rapid indicator of water quality," Appl. Environ. Microbiol., 43:430-434 [1982]; Y. Kott et al., "Bacteriophages as bacterial viral pollution indicators," Water Res., 8:165-171 [1982]; and A. H. Havelaar et al., "Factors effecting the enumeration of coliphages in sewage and sewage-polluted waters," Antonie van Leewenhoek 49:387-397 [1983]).
However, the detection limits provided by these methods are no better than those obtained with standard methods for water quality determinations based on coliform analysis. Thus, these methods do not provide a significant advantage over the traditional methods of water analysis.
In summary, the coliform group falls far short of the ideal indicator system. Coliform-free drinking water has been implicated in several waterbome outbreaks [see e.g., B. J. Dutka, "Coliforms are inadequate index of water quality," J. Environ. Health 36:39-46 [1973]). Likewise, the presence of coliforms in a particular water sample does not necessarily correlate well with the incidence of disease.
Even the enumeration of "fecal coliforms" is less than optimal, as some organisms such as Klebsiella are capable of producing positive test results. Such observations led to the development of alternative indicator organisms, including tests specific for E. coli, fecal streptococci (e.g., enterococci), Klebsiella, Clostridium perfringens, Pseudomonas aeruginosa, Bifidobacterium, Bacteroides, Aeromonas hydrophila, V. parahaemolyticus, and C. albicans, as well as other organisms commonly excreted in large numbers by healthy mammals. Notably, various opportunistic and frank pathogens uncommonly associated with waterborne transmission of disease are included in the list of indicator organisms (e.g., C. perfringens, A. hydrophila, V. parahaemolyticus, and C. albicans. Although these organisms may be useful in some settings as predictors of waterborne disease, what remains to be developed is a method for the detection and enumeration of pathogens commonly associated with waterborne diarrheal illness.
Detection and identification of Salmonella and Shigella from clinical samples has traditionally involved microbiological cultures, biochemical analyses and in some cases, serotyping methods. The same methods are used to identify suspected Salmonella or Shigella colonies isolated from clinical samples are also usually used for water, food, and other environmental samples. However, these methods are not well-suited to the unique situations associated with environmental samples, where many of the organisms present are stressed and do not perform as expected in clinical testing methods. For example, in the case of Shigella, testing problems arise due to the instability of some biochemical characteristics and antagonism of E. coli and Proteus vulgaris toward Shigella (Standard Methods, p. 927).
Despite years of regulation and testing, development of a bacterial indicator which is directly related to fecal contamination and/or the presence of pathogens which can cause waterborne disease (Pipes, p. 449) is desirable. Thus, what is needed is a cost-effective method, which is at least as sensitive and specific as traditional methods for the direct detection of pathogens present in clinical, food, water and other environmental samples.